DamID and pA-DamID
What is DamID?
DamID was initially developed by Bas van Steensel and Steve Henikoff. It is a method to map contact sites of proteins along the DNA. Just like chromatin immunoprecipitation, but based on an entirely different principle. DamID is based on the creation of a fusion protein consisting of Escherichia coli DNA adenine methyltransferase (Dam) and a chromatin protein or transcription factor of interest. Dam methylates adenines in the sequence GATC. Endogenous methylation of adenines is absent in most eukaryotes. Upon expression of the fusion protein in cultured cells or in an intact organism, Dam will be targeted to the native binding sites of the chromatin protein. This will then result in local methylation of adenine residues. Hence, the sequences near a binding site of the protein will be marked with a unique methylation tag, which can be detected using restriction enzymes (DpnI and DpnII) that are methylation sensitive. Protocols have been developed to combine this with next generation sequencing methods. DamID has been successfully used in a wide variety of model organisms and cell types.
Is it really that simple?
Almost, but there's one complication. The Dam-fusion protein is never perfectly targeted to the native binding sites — a fraction of the fusion protein molecules is inevitably diffusing around in the nucleus. This will cause considerable background methylation of non-target sites. To make things worse, some GATCs in the genome are more accessible to Dam than others, and as a result the non-specific background methylation is not homogeneous throughout the genome. Thus, one might easily mistake a non-target GATC in a very "open" chromatin region for a target of the chromatin protein.
Luckily, it turns out that one can correct for this non-targeted background methylation by measuring in a parallel control experiment the methylation levels by unfused Dam. For each GATC the methylation levels obtained with the fusion protein are then compared to the methylation levels obtained with Dam only. In practice, this comparison can be done by calculating the ratio methylation by Dam-fusion protein: methylation by Dam. We found that in this way the variation in chromatin accessibility is normalized for.
The trickiest part in the procedure
Our work in Drosophila (flies and cell lines) suggests that it is very important to keep the expression level of the Dam-fusion protein very low, to avoid saturating methylation levels. For example, both in Drosophila and mammalian cells we have used the Drosophila heat-shock promoter for expression of our Dam fusions, but in the absence of heat-shock. Under these conditions we cannot detect the fusion proteins themselves (by western blotting or immunofluorescence microscopy), but there is specific methylation of target sequences. Our interpretation is that only trace amounts of the Dam proteins are present, but that — thanks to the high enzymatic activity of Dam — this level is just right to obtain detectable but non-saturating levels of methylation. After induction the Dam-fusion proteins themselves could be detected easily, but at the same time the targeted methylation levels had reached saturation, and background methylation had become so high that no target sequences could be identified. For most applications we recommend not to drive expression of a Dam-fusion protein from the endogenous promoter of the protein of interest, as the expression will be too high.
Vectors for mammalian DamID
Our lab now only uses DamID in mammalian cells to study interactions of the genome with nuclear scaffolds such as the lamina and the nucleolus. Many other labs are using DamID to study a wide range of proteins in various model organisms. Several of our vectors are available from se can be obtained from AddGene. The basic protocol can be found here, except that we and others have adapted it for Illumina sequencing. Other labs have also published very useful DamID-seq variants and protocols. For most of our work on LADs and nucleolus interactions we now mostly use pA-DamID.
DamID in mammalian cultured cells works in principle with transient transfections. However, plasmids can accumulate in large amounts in cells after transient transfection. Since these plasmids are typically adenine-methylated, they can use up a large part of the sequencing reads. For this reason, we recommend to use either stably transfected cells, or lentivirus transduction. Various DamID vectors are available from Addgene. Feel free to contact us if you need assistance.
We have shown that it is feasible to generate single-cell DamID maps of nuclear lamina interactions. The lab of Jop Kind has pushed this technology further, and has even combined it with single-cell mRNA-seq. However, the resolution of scDamID is currently in the range of 100 kb. This is sufficient for very large chromatin domains such as LADs, but not to study proteins such as transcription factors.
Recently, we developed an antibody-based implementation of DamID to extend the toolbox. Here, a fusion protein between proteinA (pA in short) and Dam is used to target Dam to an antibody of interest. This approach is particularly useful to study effects after rapid effects, as conventional DamID requires hours to accumulate methylation signals. We applied pA-DamID to profile DNA interactions with the lamina during the cell cycle.